Malaria vaccine

ABSTRACT

The present invention provides a vaccine for preventing and/or treating  Plasmodium falciparum  infections, which comprises a polypeptide set forth in SEQ ID NO: 1 or represented by formula (1), and an adjuvant.
 
X 1 -A-B-X 2 -Y-X 3 -(Y)n-X 4 -(Y)n-X 5   (1)
 
(In the formula, X 1  represents the 1st to 7th amino acid residues in a polypeptide set forth in SEQ ID NO: 1; X 2  represents the 73th to 177th amino acid residues; X 3  represents the 178th to 258th amino acid residues; X 4  represents the 259th to 289th amino acid residues; X 5  represents the 290th to 334th amino acid residues; A represents an 8-mer repeat sequence contained in a 47-kd region of SERA polypeptide of  Plasmodium falciparum ; B represents a sequence of a serine-rich region contained in a 47-kd region of SERA polypeptide of  Plasmodium falciparum ; Y represents any one selected from A-A, A-B, and B; and n is an integer of 0 or 1.)

TECHNICAL FIELD

The present invention relates to a high-immunogenicity polypeptide thatis useful as a malaria vaccine. The present invention relates to avaccine that is useful for preventing and treating malaria parasiteinfections, and a diagnostic agent for malaria parasite infections.

BACKGROUND ART

Infectious diseases annually cause huge human and social damage indeveloping countries and elsewhere. In particular, malaria parasiteinfections annually infect 500 million people and cause the death of 2million to 3 million people; however, no effective vaccine for theprevention of this disease has been developed. Therefore, there is anurgent need to develop a malaria vaccine.

However, in spite of many vaccine clinical tests conducted over the past30 to 40 years, none of the developed vaccines showed efficacy and allefforts for vaccine development have been frustrated. In suchcircumstances, as a molecule recognized by an antibody havingantimalarial activity, SERA (serine repeat antigen) protein wasidentified in the serum of adults who have acquired protective immunity(see, for example, Non-patent Literature (NPL) 1).

Later, the development of vaccines was conducted using recombinant SERAprotein as an antigen. However, because the antibody titer of theobtained anti-SERA protein antibody is lower than that of Africans whohave gained protection (immunity) against malaria parasite infections,further improvement has been desired.

CITATION LIST Non-Patent Literature

-   NPL 1: Bzik D J, Li W B, Horii T, Inselburg J. Mol Biochem    Parasitol. 1988 September; 30(3): 279-88

SUMMARY OF INVENTION Technical Problem

A primary object of the present invention is to provide ahigh-immunogenicity polypeptide that is useful as a malaria vaccine, andto provide the use of the polypeptide as a vaccine against malariaparasite infections. Another object of the present invention is toprovide an antibody to the polypeptide, and the use of the antibody orthe polypeptide as a diagnostic agent for malaria parasite infections.

Solution to Problem

The present inventors previously identified that an 8-mer repeat regionand/or a serine-rich region, which are present on the N-terminus of aSERA (serine repeat antigen) polypeptide, are protective epitopes ofantibodies to Plasmodium falciparum.

Now the present inventors have found a polypeptide that has excellentimmunogenicity and is useful as a malaria vaccine. In addition, thepresent inventors have found an optimal adjuvant for enhancing theimmunogenicity of a malaria vaccine. The inventors conducted furtherresearch based on these findings and accomplished the present invention.

The present invention includes the following embodiments.

(I) Polypeptide Useful as a Malaria Vaccine, and Polynucleotide Encodingthe Polypeptide

-   (I-1) A polypeptide represented by the following formula (1):    X₁-A-B-X₂-Y-X₃-(Y)n-X₄-(Y)n-X₅  (1)    (wherein-   X₁ represents the 1st to 7th amino acid residues in a polypeptide    set forth in SEQ ID NO: 1;-   X₂ represents the 73th to 177th amino acid residues of the    polypeptide;-   X₃ represents the 178th to 258th amino acid residues of the    polypeptide;-   X₄ represents the 259th to 289th amino acid residues of the    polypeptide;-   X₅ represents the 290th to 334th amino acid residues of the    polypeptide;-   A represents an 8-mer repeat sequence contained in a 47-kd region of    SERA polypeptide of Plasmodium falciparum;-   B represents a sequence of a serine-rich region contained in a 47-kd    region of SERA polypeptide of Plasmodium falciparum;-   Y represents any one selected from A-A, A-B, and B; and-   n represents an integer of 0 or 1).-   (I-2) The polypeptide according to (I-1), wherein, in formula (1), Y    is A-B or B.-   (I-3) The polypeptide according to (I-1), wherein, in formula (1), A    is an amino acid sequence selected from SEQ ID NOS: 2 to 8, and B is    an amino acid sequence set forth in SEQ ID NO: 9.-   (I-4) The polypeptide according to (I-1), wherein the polypeptide    represented by formula (1) is a polypeptide represented by one of    the formulas (2) to (6):    X₁-A₁-B-X₂-(A₂-B)-X₃-X₄-X₅  (2)    X₁-A₁-B-X₂-(A₂-B)-X₃-(A₃-B)-X₄-X₅  (3)    X₁-k-B-X₂-(A₂-B)-X₃-(A₃-B)-X₄-(A₄-B)-X₅  (4)    X₁-A₁-B-X₂-(A₂-As)-X₃-(A₃-A₆)-X₄-(A₄-A₇)-X₅  (5)    X₁-k-B-X₂-(B)-X₃-(B)-X₄-(B)-X₅  (6)

(wherein

-   X₁ represents the 1st to 7th amino acid residues in the polypeptide    set forth in SEQ ID NO: 1;-   X₂ represents the 73rd to 177th amino acid residues of the    polypeptide;-   X₃ represents the 178th to 258th amino acid residue of the    polypeptide;-   X₄ represents the 259th to 289th amino acid residue of the    polypeptide;-   X₅ represents the 290 to 334th amino acid residue of the    polypeptide;-   A₁ to A₇ represent amino acid sequences set forth in SEQ ID NOS: 2    to 8, respectively; and-   B represents an amino acid sequence set forth in SEQ ID NO: 9).-   (I-5) The polypeptide according to any one of (I-1) to (I-4),    comprising an amino acid sequence set forth in one of SEQ ID NOS: 10    to 14.-   (I-6) A polynucleotide encoding at least one of the polypeptides    according to (I-1) to (I-5).    (II) Vaccine for Preventing and/or Treating a Plasmodium falciparum    Infection-   (II-1) A vaccine for preventing and/or treating a Plasmodium    falciparum infection, comprising as an active ingredient at least    one of the polypeptides according to (I-1) to (I-5).-   (II-2) The vaccine according to (II-1), further comprising at least    one adjuvant selected from the group consisting of aluminium    hydroxide gel, K3 (K-type CpG adjuvant), D35 (D-type CpG adjuvant),    and sHZ (synthetic hemozoin adjuvant).-   (II-3) The vaccine according to (II-1) or (II-2), further comprising    a ligand having innate immune-stimulatory activity.-   (II-4) The polypeptide according to any one of (I-1) to (I-5), which    is used as a vaccine for preventing and/or treating a Plasmodium    falciparum infection.-   (II-5) A combination of the polypeptide according to one of (I-1) to    (I-5) with at least one adjuvant selected from the group consisting    of aluminium hydroxide gel, K3 (K-type CpG adjuvant), D35 (D-type    CpG adjuvant), and sHZ (synthetic hemozoin adjuvant), the    combination being used as a vaccine for preventing and/or treating a    Plasmodium falciparum infection.-   (II-6) A combination of the polypeptide according to one of (I-1) to    (I-5) with at least one adjuvant selected from the group consisting    of aluminium hydroxide gel, K3 (K-type CpG adjuvant), D35 (D-type    CpG adjuvant), and sHZ (synthetic hemozoin adjuvant), and a ligand    having innate immune-stimulatory activity, the combination being    used as a vaccine for preventing and/or treating a Plasmodium    falciparum infection.-   (II-7) A vaccine for preventing and/or treating a Plasmodium    falciparum infection, comprising the polypeptide set forth in SEQ ID    NO: 1 and an adjuvant.-   (II-8) The vaccine according to (II-7), wherein the adjuvant is at    least one member selected from the group consisting of K3 (K-type    CpG adjuvant), D35 (D-type CpG adjuvant), and sHZ (synthetic    hemozoin adjuvant), or a combination of at least one of these    adjuvants with aluminium hydroxide gel.-   (II-9) The vaccine according (II-7) or (II-8), further comprising a    ligand having innate immune-stimulatory activity.-   (II-10) A combination of a polypeptide set forth in SEQ ID NO: 1    with an adjuvant, the combination being used as a vaccine for    preventing and/or treating a Plasmodium falciparum infection.-   (II-11) A combination of a polypeptide set forth in SEQ ID NO: 1, an    adjuvant, a ligand having innate immune-stimulatory activity, the    combination being used as a vaccine for preventing and/or treating a    Plasmodium falciparum infection.-   (II-12) The combination according to (II-10) or (II-11) wherein the    adjuvant is at least one member selected from the group consisting    of K3 (K-type CpG adjuvant), D35 (D-type CpG adjuvant), and sHZ    (synthetic hemozoin adjuvant), or a combination of at least one of    these adjuvants with aluminium hydroxide gel.    (III) Diagnostic Agent for a Plasmodium falciparum Infection-   (III-1) An antibody to the polypeptide according to one of (I-1) to    (I-5).-   (III-2) The antibody according to (III-1) having affinity for    polypeptides set forth in SEQ ID NO: 23 to 37.-   (III-3) The antibody according to (III-1) having affinity for    polypeptides set forth in SEQ ID NO: 23 to 25.-   (III-4) A diagnostic agent for a Plasmodium falciparum infection,    comprising, as an active ingredient, the polypeptide according to    one of (I-1) to (I-5), or the antibody according to one of (III-1)    to (III-3).

Advantageous Effects of Invention

-   (i) The polypeptide represented by formula (1) (hereinafter    sometimes simply referred to as “polypeptide (1)”) has high    immunogenicity and can maintain high anti-SE36 antibody titers over    a long period of time when used as a vaccine. Accordingly, immunity    against malaria parasites can be acquired, and the acquired immunity    can be maintained for a long period of time.-   (ii) According to the polypeptide (1), sufficient immunity can be    acquired with a dose that is 1/10th to 1/100th the amount of an    antigen polypeptide required to be used to produce an antibody to    malaria parasites in vivo. Due to such a small dose, the    polypeptide (1) is useful from an economic viewpoint as well.-   (iii) The use of the polypeptide (1) as a vaccine can induce an    antibody to a protective epitope. Therefore, the polypeptide (1) of    the present invention can be used as a malaria vaccine having high    immunogenicity (particularly a Plasmodium falciparum malaria    vaccine).-   (iv) According to the present invention, a combination of the    polypeptide set forth in SEQ ID NO: 1 (hereinafter sometimes    referred to as “polypeptide (2)” or “original SE36 polypeptide”)    with an optimal adjuvant can be provided. Although the    polypeptide (2) itself also has higher antigenicity than a natural    SERA polypeptide, a combination of the polypeptide (2) with specific    adjuvant or adjuvants can exhibit more remarkable excellent    antigenicity. Accordingly, the combined use of the polypeptide (2)    with specific adjuvant(s) (e.g., a combination of the    polypeptide (2) with a human TLR9 ligand adjuvant, or with aluminium    hydroxide gel and a human TLR9 ligand adjuvant, particularly K3    (K-type CpG adjuvant), can induce even higher anti-SE36 antibody    titers.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1-1 shows the amino acid sequences of polypeptide (2) (originalSE36 polypeptide) and polypeptides (1) (SE36-1 to SE36-3).

FIG. 1-2 shows the amino acid sequences of polypeptides (1) (SE36-4 toSE36-5) and a negative SE36 polypeptide.

FIG. 1-3 shows the base sequence of a nucleotide encoding the amino acidsequence of the polypeptide (2) (original SE36 polypeptide).

FIG. 1-4 shows the base sequence of a nucleotide encoding the amino acidsequence of the polypeptide (1) (SE36-1).

FIG. 1-5 shows the base sequence of a nucleotide encoding the amino acidsequence of the polypeptide (1) (SE36-2).

FIG. 1-6 shows the base sequence of a nucleotide encoding the amino acidsequence of the polypeptide (1) (SE36-3).

FIG. 1-7 shows the base sequence of a nucleotide encoding the amino acidsequence of the polypeptide (1) (SE36-4).

FIG. 1-8 shows the base sequence of a nucleotide encoding the amino acidsequence of the negative SE36 polypeptide.

FIG. 1-9 shows the amino acid sequence of an epitope mappingpolypeptide.

FIG. 2 shows the location and positional relationship between the 8-merrepeat region and the serine-rich region in the polypeptide (2)(original SE36 polypeptide), polypeptides (1) (SE36-1 to 36-5polypeptides), and negative SE36 polypeptide.

FIG. 3 shows the results of measurement of antibody titers of the wholeIgG against SE36 after inoculation of the polypeptide (2) (original SE36polypeptide), polypeptides (1) (SE36-1 to SE36-5 polypeptides), andnegative SE36 polypeptide.

FIG. 4 shows the results of epitope mapping of the polypeptide (2)(original SE36 polypeptide), and polypeptides (1) (SE36-1 to SE36-5polypeptides) (Test Example 2). In FIG. 4, 1 to 15 on the abscissa showthe polypeptides used for mapping 15 epitopes set forth in SEQ ID NO: 23to 37.

FIG. 5 shows the results of antibody induction by combined use of thepolypeptide (2) (original SE36 polypeptide) and adjuvant(s) (aluminiumhydroxide gel (Alum) alone, K3 alone, or aluminium hydroxide gel (Alum)and K3).

FIG. 6 is a graph showing changes over time in antibody titers of thewhole IgG measured after combined use of various adjuvants (aluminiumhydroxide gel (Alum); aluminium hydroxide gel (Alum) and K3; aluminiumhydroxide gel (Alum) and D35; and aluminium hydroxide gel (Alum) andsHZ) with the polypeptide (2) (original SE36 polypeptide) versus the useof the polypeptide alone (2) (original SE36 polypeptide).

FIG. 7 is a graph showing the results of epitope mapping in Test Example3. In FIG. 7, the polypeptides of SEQ ID NO: 23 to 37 used for mapping15 epitopes are shown as 1 to 15 on the abscissa. Used as adjuvants forthe polypeptide (2) were aluminium hydroxide gel (Alum) only (top-leftfigure), aluminium hydroxide gel and K3 (Alum+K3) (K-type CpG adjuvant)(top-right figure), aluminium hydroxide gel and D35 (D-type CpGadjuvant) (Alum+D35) (bottom-left figure), or aluminium hydroxide geland sHZ (synthetic hemozoin adjuvant) (Alum+sHZ) (bottom-right figure).

FIG. 8 shows the 14-day measurement results of the number of malariaparasite-infected red blood cells in the blood (parasitemia (%))measured after immunization of squirrel monkeys with the polypeptide (2)and aluminium hydroxide gel (Alum) (n=2) (left figure); polypeptide (2)(SE36), aluminium hydroxide gel (Alum), and K3 (n=3) (center figure);and aluminium hydroxide gel (Alum) and K3 (n=2) (right figure), aftersubsequent inoculation with a malaria parasite. In FIG. 8, “dt”indicates ethical death of squirrel monkeys.

DESCRIPTION OF EMBODIMENTS

1. SERA Polypeptide Variant

The polypeptides (1) and (2) of the present invention, which are usefulas vaccines for malaria parasite infections, are described below indetail. Herein, the polypeptides (1) and (2) may be collectivelyreferred to as “the polypeptide of the present invention”.

(1-1) SERA polypeptide

SERA (serine-repeat antigen) is a protein antigen with a molecularweight of 115 kd consisting of 989 amino acids in total and expressed bya Pf gene at the intra-erythrocyte stage. The structure of SERA consistsof 3 domains, i.e., 47 kd-50 kd-18 kd, in order of the N-terminal to theC-terminal direction. The SERA working as a precursor for these domainsis expressed by 4 exons comprising a total of 5868 bases, processed andcleaved at the intra-erythrocyte stage during merozoite release to yieldthe above-described domains (Molecular and Biochemical Parasitology, 86,pp. 249-254, 1997; and Experimental Parasitology, 85, pp. 121-134,1997). In addition, the data on the full length of the SERA gene (DNA)and the amino acid sequence encoded by this gene are open to the publicand available from GenBank (Accession Number: J04000;www.ncbi.nlm.nih.gov). The N-terminal region of SERA (hereinafterreferred to as “47 kd region”) consists of 382 amino acids in total. Thehomology search between the Pf strains relative to the sequenceindicates that SERA is varied since in some regions there is amino aciddeletion or addition, or amino acid variation (non-synonymoussubstitution) at about 20 positions (Molecular and BiochemicalParasitology, supra; and Experimental Parasitology, supra).

(1-2) Polypeptide (2)

Polypeptide (2) is the original SE36 polypeptide set forth in SEQ ID NO:1.

The polypeptide (2) is derived from an SE47′ antigen (Vaccine, 14, pp.1069-1076, 1996; hereinafter simply referred to as “SE47′”), which isbased on a 47-kd region of SERA of the above-described Honduras-1 strainof Plasmodium falciparum (Pf) (hereafter simply referred to as“Hond-1”). The polypeptide (2) consists of a total of 334 amino acids,in which the sequence starts from the N-terminal methionine (the 1stamino acid) of 382 amino acids constituting 47 kd of SERA of Hond-1, andin the ordinal number towards the C-terminal, the 16th amino acid codon(aspartic acid) is substituted with an initiation codon (methionine),and a translation stop codon is inserted after the 382nd amino acid(glutamic acid), and further the 33 polymerized serine residues (193rdto 225th serines) occupying the serine repeat region are deleted. Theamino acid sequence of the polypeptide (2) is specifically shown as SEQID NO: 1 in FIG. 1-1. The polypeptide (2) set forth in SEQ ID NO: 1 hashigher immunogenicity than the natural SERA polypeptide.

(1-3) Polypeptide (1)

Polypeptide (1) is an improved SE36 polypeptide obtained by furtherimproving the polypeptide set forth in SEQ ID NO: 1 (2) to have higherimmunogenicity.

The polypeptide (1) is represented by the following formula (1):X₁-A-B-X₂-Y-X₃-(Y)n-X₄-(Y)n-X₅  (1)wherein

-   X₁ represents the 1st to 7th amino acid residues in the polypeptide    set forth in SEQ ID NO: 1;-   X₂ represents the 73th to 177th amino acid residues of the    polypeptide;-   X₃ represents the 178th to 258th amino acid residues of the    polypeptide;-   X₄ represents the 259th to 289th amino acid residues of the    polypeptide;-   X₅ represents the 290th to 334th amino acid residues of the    polypeptide;-   Y represents any one selected from A-A, A-B, and B; and-   n represents an integer of 0 or 1.

In formula (1), A represents an 8-mer repeat sequence contained in a47-kd region of SERA polypeptide of Plasmodium falciparum. In thepresent invention, any sequence that corresponds to the 8-mer repeatsequence can be used as sequence A, even if there is amino acidvariation (non-synonymous substitution) among Pf strains. Examples ofamino acid sequences that can be preferably used as sequence A includethe following specific sequences A₁ to A₇ (shown with the N-terminus atthe left).

(SEQ ID NO: 2) A₁: TGESQTGNTGGGQAGNTVGDQAGSTGGSPQGSTGASQPGS(SEQ ID NO: 3)A₂: TGESQTGNTGGGQAGNTGGDQAGSTGGSPQGSTGASPQGSTGASPQGSTGASQPGS(SEQ ID NO: 4) A₃: TGESQTGNTGGGQAGNTVGDQAGNTVGDQAGSTGGSPQGSTGASQPGS(SEQ ID NO: 5) A₄: TGESQTGNTGGGQAGNTGGGQAGNTVGDQAGSTGGSPQGSTGASQPGS(SEQ ID NO: 6) A₅: TGESQTGNTGGGQVGNTGGGQAGSTGGSPQGSTGASQPGSSEPSNPVS(SEQ ID NO: 7) A₆: TGESQTGNTGGGQAGNTVGGQAGNTGGGQAGNTGGDPQGSTGGSQPGS(SEQ ID NO: 8)A₇: TGESQTGNAGGGQAGNTVGDQAGSTGGSPQGSTGASPQGSTGASPQGSTGASQPGS.

In formula (1), A linked to X₁ is preferably A₁ (SEQ ID NO: 2).

In formula (1), B represents the amino acid sequence of a serine-richregion in the 47-kd region of the SERA polypeptide of Plasmodiumfalciparum. In the present invention, any sequence that corresponds tothe amino acid sequence of the serine-rich region can be used assequence B, even if there is amino acid variation (non-synonymoussubstitution) among Pf strains. Examples of amino acid sequences thatcan be preferably used as sequence B include the following specificsequence (shown with the N-terminus at the left)

(SEQ ID NO: 9) B: SEPSNPVSSGHSVSTVSVSQTSTSS.

In formula (1), A and B can be suitably selected from the above specificsequences (SEQ ID NO: 2 to 8, and SEQ ID NO: 9) and used in combination.The amino acid sequence A may be any one selected from the above aminoacid sequences shown as A₁ to A₇. The amino acid sequence of onepolypeptide may contain, as the amino acid sequence A, two or more ofthe same amino acid sequence selected from A₁ to A₇, or any combinationof different amino acid sequences selected from A₁ to A₇.

Y may be suitably selected from A-A, A-B, and B. Preferably, Y is eitherA-B or B.

Examples of preferable polypeptides (1) in the present invention includepolypeptides (1) set forth in SEQ ID NO: 10 to 14, i.e., SE36-1 toSE36-5. FIG. 2 shows a schematic diagram thereof. As in formula (1),SE36-1 to SE36-5 can be represented by the following formulas:Formula: X₁-A-B-X₂-Y-X₃-(Y)n-X₄-(Y)n-X₅SE36-1: X₁-A₁-B-X₂-(A₂-B)-X₃-(Y)0-X₄-(Y)0-X₅SE36-2: X₁-A₁-B-X₂-(A₂-B)-X₃-(A₃-B)-X₄-(Y)0-X₅SE36-3: X₁-A₁-B-X₂-(A₂-B)-X₃-(A₃-B)-X₄-(A₄-B)-X₅SE36-4: X₁-A₁-B-X₂-(A₂-A₅)-X₃-(A₃-A₆)-X₄-(A₄-A₇)-X₅SE36-5: X₁-A₁-B-X₂-(B)-X₃-(B)-X₄-(B)-X₅  (1)(in the above formulas, (Y)0 means that there is no amino acid residuecorresponding to Y; that is, (Y)0 indicates a single bond).

Among SE36-1 to SE36-5, SE36-3 to SE36-5 are preferable, SE36-3 andSE36-5 are more preferable, and SE36-3 is particularly preferable.

(1-4) Synthesis of the Polypeptide of the Present Invention

The polypeptide of the present invention can be synthesized by knownmethods, and the synthesis method is not particularly limited. Forexample, the polypeptide (2) can be synthesized by the following method.After a DNA fragment (hereinafter sometimes referred to as “SE36 gene(DNA)”) encoding the polypeptide (2) is synthesized and cloned, anexpression vector for the synthesized gene clone is constructed. Thevector is transfected into a host (for example, Escherichia coli), andthe obtained transformant is cultured.

The expression efficiency of SE36 gene (DNA) having native Pf codons inEscherichia coli is low. Therefore, when E. coli is used as a host, itis desirable to convert all naturally occurring Pf codons encoding theamino acid sequence of the polypeptide of the present invention intoEscherichia coli codons to achieve efficient production of thepolypeptide as in the present invention.

Although the synthesis of the polypeptide (2) is explained below, thepolypeptide (1) can also be synthesized in a similar manner.

Synthesis and Cloning of SE36 Gene (DNA)

A theoretical base sequence of an SE36 gene (DNA) of Pf (SE36 genehaving Pf codons) and an amino acid sequence coded thereby are availablefrom well-known gene database publication organizations, such as DDBJ,GenBank, and EMBL on the Internet. The amino acid sequence of thepolypeptide (2) is encoded by the nucleotide sequence set forth in SEQID NO: 16 (FIGS. 1 to 3). Accordingly, SE36 gene (DNA) encoding theamino acid sequence (SEQ ID NO: 1) of the polypeptide (2) is synthesizedbased on SEQ ID NO: 16. For example, DNA encoding the amino acidsequence of the polypeptide (1) is synthesized based on the nucleotidesequences set forth in SEQ ID NO: 17 to 22 that encode the amino acidsequences (SEQ ID NO: 10 to 14) of SE36-1 to SE36-5.

Theoretical conversion of Pf codons into Escherichia coli codons may beconducted referring to, for example, the codon usage database of GenBankand publications of the present inventors (the above-mentioned Vaccine;and Molecular Biochemistry of Parasitology, 63, 265-273, 1994). In theconversion into Escherichia coli codons, care should be taken to notdamage the antigenicity due to non-synonymous substitution of aminoacids except for substitution of the N-terminal amino acid with aninitiation codon Met, because the native amino acid sequence ofnaturally occurring Pf is considered to be important.

In the synthesis of DNA, a commercially available DNA synthesizer, suchas a DNA/RNA synthesizer (Applied Biosystems Model 392, a product of PECo., USA) or an ASU-102U DNA synthesizer (a product of Biosset Ltd.,USA) can be used. By using such a synthesizer, sense (+) and antisense(−) DNA fragments, in which about 100 to about 200 nucleotides arepolymerized, are separately synthesized, and each DNA fragment thussynthesized is purified, for example, by polyacrylamide electrophoresis.Subsequently, a complementary strand (pair) of the purifiedsingle-stranded DNA fragment is annealed to give a syntheticdouble-stranded DNA fragment.

In cloning of the synthetic double-stranded DNA fragment, a known orcommercially available cloning vector of which the host is Escherichiacoli (various vectors disclosed in “Cloning Vectors: A LaboratoryManual”, 1-1 to 1-D-1-8, P. H. Pouwels, et al., Elsevier 1988), forexample, a combination of a plasmid pBluescript II SK and E. coliXL1-BLue (Stratagene, USA) may be used. In such cloning, for example, arestriction enzyme-digested fragment of the above-mentioned DNA fragmentis inserted into the restriction enzyme sites of a vector digested withthe same enzyme, and the thus constructed vector is transferred to ahost to give a transformant clone. Subsequently, each clone containingthe double-stranded DNA fragment is amplified by culturing thetransformant, and each base sequence can be determined by means of thechain terminator method (dideoxy method) or the Maxam-Gilbert method. Inthis procedure, a commercially available DNA sequencer, e.g., ABI PRISM3700 (a product of PE Co., USA), may be used. Based on the results,approximately 5 to 10 clones of double-stranded DNA fragments coveringthe full length of SE36 gene DNA are selected.

The cloning of the full length of an SE36 gene (DNA) may be achieved bysequentially ligating the aforementioned double-stranded DNA fragmentstogether. For example, when 8 clones (8 pairs) are obtained in the aboveoperation, these double-stranded DNA fragments can be ligated one by oneto give a full-length SE36 gene DNA. Then, the full-length DNA is clonedin the same manner as mentioned above. In carrying out the ligation, itis desirable to introduce a cohesive site for ligation by restrictionenzymes at both ends of each fragment of the double-stranded DNAs. Insuch introduction, the codon base sequence(s) has to be adjusted so thatthe amino acid sequence of the native Pf is not changed. In thepolypeptide (1), the cohesive site for ligation by restriction enzymesmay remain in the ligation site of the full-length DNA, or may betranslated as is and be present in an amino acid sequence.

Construction of Expression System of SE36 Gene

In producing an expression vector for an SE36 gene (DNA), a known orcommercially available expression vector of which the host isEscherichia coli (variously disclosed in “Cloning Vectors: A LaboratoryManual”; supra), such as, a combination of plasmid pET-3a andEscherichia coli BL21 (DL3) pLysS or Escherichia coli BL21 (DL3) pLysE(a product of Stratagene, USA) may be used. In producing such a vector,for example, a restriction enzyme-digested fragment containing SE36 gene(DNA) is cut out from the above cloning vector and then inserted intothe restriction enzyme site of a vector cleaved with the same enzyme.Alternatively, the expression vector for an SE36 gene may also beprepared from pET-SE47′ (Vaccine, supra). In this case, for example, thewhole or partial region that encodes the serine repeat region is cleavedby a restriction enzyme from pET-SE47′ or an SE47′ synthetic genecontained therein, and the expression vector for an SE36 gene isprepared in the same manner as above. Subsequently, the resultingexpression vector is transferred to a host to give a transformant. Fromsuch transformants, the most appropriate one as an expression system formass production of the polypeptide SE36 can be screened in view ofindustrial applicability.

Mass production of the polypeptide of the present invention is achievedby culturing the aforementioned transformant of Escherichia coli. Inview of enhancement of productivity of the polypeptide of the invention,in such a culture, it is possible to improve or reform a manipulationsuch as the use of an inducer, e.g., IPTG(isopropyl-1-thio-β-D-galactopyranoside), avoidance of cataboliterepression, culture medium composition, culture temperature and period,removal of proteases in host cells, and the like. These may be achievedby alteration of an expression vector, change of a promoter or hostorganism, etc.

Purification of Polypeptide

When the polypeptide of the present invention is secreted outside thecells, the culture solution after removal of the cells from theaforementioned transformant culture is used as a starting material forextraction of the SE36 polypeptide. When SE36 is accumulated in thecells, the cells may be recovered from the culture, for example, bycentrifugation, filtration, etc., and extracted to give the above SE36polypeptide. At the first step of extraction, the cells may be destroyedby digestion with an enzyme, destruction with osmotic pressure, suddenpressure and decompression, sonication, use of various homogenizers,etc. The destroyed cells are then fractionated by physical means such aslow-speed centrifugation, ultra-centrifugation, filtration, a molecularsieve, membrane concentration, etc.; or by chemical means such as aprecipitating agent, a solubilizing agent, an adsorbent and desorptionagent, a dispersing agent, etc.; or by physicochemical means such aselectrophoresis, column chromatography, a support, dialysis,salting-out, etc. These techniques may be used in combination. Inapplying these techniques, physicochemical conditions, such astemperature, pressure, pH, and ion strength, can be suitably set.

Polypeptide of the Present Invention and Confirmation of itsAntigenicity

Detection and size confirmation of the obtained polypeptide of thepresent invention may be achieved, for example, by determination ofsedimentation coefficient, molecular sieve, SDS-polyacrylamideelectrophoresis, etc. Antigenicity of the polypeptide of the inventionmay be confirmed by means of an antigen-antibody reaction using apolyclonal or monoclonal antibody to SERA 47 kd, such as, Western blotanalysis, ELISA, agglutination reaction, fluorescent antibody technique,radioimmunoassay, and the like. In addition, immunogenicity of thepolypeptide of the present invention and the ability of an anti-SE36antibody to inhibit Pf parasite growth can be confirmed, for example, bymeans of an antigen-antibody reaction using the serum of a patientsuffering from Pf malaria or an experimental small animal immunized withsaid polypeptide, such as a rat or mouse, or by growth inhibition (i.e.,a neutralization reaction) of Pf merozoites within erythrocytes, ordetermination of the blood Pf number in an anti-SE36 antibody carrier.

2. Malaria Vaccine

The present invention provides a malaria vaccine comprising thepolypeptide of the present invention. Vivax malaria, malariae malaria,ovale malaria, and falciparum malaria are known as malaria parasiteinfections. The vaccine of the present invention is suitable for use asa falciparum malaria vaccine because it has remarkably high specificityas a falciparum malaria antigen. However, the use of the vaccine is notlimited thereto, and the vaccine can be used for other malaria parasiteinfections.

An example method for preparing a vaccine using the polypeptide (2) isdescribed below. However, a vaccine can also be prepared by using thepolypeptide (1) in a similar manner.

(2-1) Preparation of a Vaccine

As an antigen, the polypeptide (2) purified above is dissolved in asolvent, such as isotonic PBS (phosphate buffer saline), to give avaccine stock solution.

The above antigen for vaccine may be immobilized with a conventionalinactivating agent to stabilize the steric structure. Examples ofinactivating agents that can be used include formalin, phenol, glutaricdialdehyde, β-propiolactone, and the like, which may be added before orafter preparation of the vaccine stock solution. When formalin is used,the amount to be added is about 0.005 to 0.1% (v/v), the inactivationtemperature is about 4 to 38° C., and the inactivation period is about 5to 180 days. If the antigenicity is damaged by inactivation, ingenuityis required to moderate the inactivation conditions. Such moderation maybe achieved, for example, by reduction of the amount of inactivatingagent used, addition of a neutral or basic amino acid, lowering of theinactivation temperature, etc. Free formaldehyde remaining unchangedafter the inactivation step may be, if required, neutralized withaddition of an equivalent of sodium hydrogen sulfite or removed bydialysis.

In order to induce mucous or local immunity by oral or nasal inoculationof a vaccine, the polypeptide (2) (i.e., antigen) may be processed ormodified. For this purpose, a drug delivery system (DDS) techniqueusing, for example, liposome, emulsion, microcapsules, micro-spheres,polylactic acid, polyglycolic acid, etc., may be applied. Thepreparation thus obtained is used as a vaccine stock solution in thesubsequent step.

The vaccine stock solution is diluted, for example, with theabove-mentioned PBS to adjust the amount of the antigen in the vaccineso that antibody production is induced and immunity is established. Inthis process, it is possible to add a stabilizer for increasing the heatresistance of the vaccine and to add an adjuvant as an auxiliary forenhancing antigenicity. As a stabilizer, for example, sugars or aminoacids may be used. Mineral oil, vegetable oil, alum, aluminum compounds(e.g., aluminium hydroxide gel), bentonite, silica, muramyl dipeptidederivatives, thymosin, interleukin, etc., may be used as an adjuvant.Examples of adjuvants that can be preferably used include, as describedbelow, aluminium hydroxide gel and human TLR9 ligand adjuvants. Specificexamples of human TLR9 ligand adjuvants include K3 (K-type CpGadjuvant), D35 (D-type CpG ODN adjuvant), and sHZ (synthetic hemozoinadjuvant).

Subsequently, the resulting vaccine is dispensed into vials in anappropriate amount, such as in vials of about 1 to 20 ml, and the vialsare tightly closed or sealed for use as vaccine preparations. Suchvaccine preparations may be used in a liquid state, or formed into drypreparations by lyophilization after dispensing and used.

(2-2) Assay of Vaccines

Assay of vaccines, which is related to production process control andquality control, is conducted in accordance with the Japanese Rules for“Minimum Requirements for Biological Products” based on thePharmaceutical Affairs Law (Law No. 145 enacted in 1960), Article 42,Section 1; WHO recommendation on “Requirements for BiologicalSubstances” (WHO Technical Report Series (TRS), No. 889, pp. 105 to 111,1999), etc. A malaria vaccine has not yet been put to practical use, andthere is no standard for pharmaceutical preparations. The assay,therefore, may be conducted in accordance with a standard for ananalogous vaccine, such as the variety of rules on safety and efficacyas described in WHO recommendation on “Requirements for Hepatitis BVaccines Made by Recombinant DNA Techniques” (the aforementioned TRS,No. 786, 1898, and No. 889, 1999), and “Requirements for JapaneseEncephalitis Vaccine (Inactivated) for Human Use” (the aforementionedTRS, No. 771, 1988), etc. For example, the assay for sterilization,denial of abnormal toxicity, protein content, purity, hydrogen ionconcentration, confirmation of antigens, antigenic polypeptides, and thelike may be conducted in accordance with the rules for a variety ofrequired or recommended tests. A product lot that has passed all of theabove tests may be put to practical use as a qualified malaria vaccinepreparation.

(2-3) How to Use the Vaccine

The vaccine can be inoculated according to methods common in the art,and the method of vaccine inoculation is not particularly limited. Forexample, the vaccine can be subcutaneously inoculated at a dose of about0.25 to 0.5 ml. Such inoculation may be preferably performed 1 to 3times at intervals of about 2 to 4 weeks.

3. Adjuvants and Vaccine Comprising a Combination of the Adjuvants

(3-1) In preparation of the vaccine, the polypeptide (1) may be usedtogether with a combination of known adjuvants. Such adjuvants are notparticularly limited, and any adjuvant that can enhance the immunizingeffect of the present invention can be used. Examples of usableadjuvants include aluminium hydroxide gel and human TLR9 ligandadjuvants such as K3 (K-type CpG adjuvant), D35 (D-type CpG ODNadjuvant), and sHZ (synthetic hemozoin adjuvant). In addition to theseadjuvants, ligands having innate immune-stimulatory activity can also beused as adjuvants.

In the present invention, adjuvants may be used singly or in combinationof two or more selected from such adjuvants. Aluminium hydroxide gel hasthe property of forming an insoluble antigen-adjuvant complex. Due tolocal accumulation of this antigen-adjuvant complex, combined use ofaluminium hydroxide gel with other adjuvant(s) as mentioned above ispreferable.

(3-2) In preparation of the vaccine, the polypeptide (2) may also beused together with known adjuvants as exemplified above. Examples ofadjuvants that can be preferably used include K3 (K-type CpG adjuvant),D35 (D-type CpG ODN adjuvant), and sHZ (synthetic hemozoin adjuvant),which are human TLR9 ligand adjuvants. However, when the polypeptide (2)is used with a specific combination of adjuvants, particularly excellenteffects can be achieved.

Examples of combinations of adjuvants with which the polypeptide (2) canprovide particularly excellent immunogenicity include, in addition tocombinations of the above-mentioned humans TLR9 ligand adjuvants, (a) acombination of aluminium hydroxide gel and K3 (K-type CpG adjuvant), (b)a combination of aluminium hydroxide gel and D35 (D-type CpG adjuvant),and (c) a combination of aluminium hydroxide gel and sHZ (synthetichemozoin adjuvant). Among these, (a) a combination of aluminiumhydroxide gel and K3 (K-type CpG adjuvant) can maintain high anti-SE36antibody titers for a long period of time. Accordingly, use of thepolypeptide (2) with a combination of specific adjuvants as mentionedabove is effective as a malaria vaccine.

4. Diagnostic Agent

The antibody to the polypeptide (1) or (2) can be used for the diagnosisof malaria parasite infections. More specifically, the present inventioncan provide a diagnostic agent for malaria parasite infections,comprising an antibody to the polypeptide (1) or (2) as an activeingredient. The antibody to the polypeptide (1) or (2) can be obtained,for example, by intraperitoneally, subcutaneously, or intramuscularlyinoculating the polypeptide (1) or (2) to an animal, such as a rabbit,guinea pig, or mouse, to generate an antibody and isolate the antibodyfrom the serum of the animal. Such an antibody can be used for detectionof an antigen. If the antigen is detected in a sample, for example,serum, of a patient suspected of a malaria parasite infection, thepatient can be diagnosed as being infected with a malaria parasite.

An anti-SE36 antibody can be detected by a precipitation reaction,agglutination reaction, neutralization reaction, fluorescent antibodytechnique, enzyme immunoassay, radioimmunoassay, or the like, using thepolypeptide (1) or (2) as an antigen and using the serum, etc., of apatient suspected of a malaria parasite infection as a sample.Accordingly, if the antigen is detected in the sample, the patient isdiagnosed as being infected with a malaria parasite.

The antigen and antibody used in diagnosis according to the presentinvention may be diluted with a solvent, such as the above-mentionedPBS, so that the content of the antigen and antibody in the diagnosticagent becomes the amount necessary for the antigen/antibody reaction.

5. Method for Preventing and/or Treating Malaria Parasite Infections

The vaccine of the present invention has high antibody titers asmentioned above. Therefore, malaria parasite infections can beeffectively prevented by pre-administering the vaccine to a subject,such as person in need of prevention against malaria parasite infection.

Because the vaccine of the present invention is particularly effectiveagainst Plasmodium falciparum infections, Plasmodium falciparuminfections can be effectively treated by administering the vaccine to apatient infected with Plasmodium falciparum. Thus, the present inventionfurther provides a method for preventing and/or treating malaria using amalaria vaccine. The malaria vaccine preparation method, dosage, etc.,are as described above.

As described above, administration of the polypeptide (1) or (2) of thepresent invention together with adjuvants as mentioned above can furtherenhance malaria preventive and/or therapeutic effects.

EXAMPLES

The present invention is described below in more detail with referenceto Test Examples. However, the scope of the invention is not limited tothese Examples.

Test Example 1

The present inventors previously concluded from the results of epitopemapping directed to SE36 (SEQ ID NO: 1) that either one of or both ofthe 8-mer repeat and serine-rich regions that are present in theN-terminal region are important as protective epitopes. (The documentthat discloses this will be described here.)

Based on the above finding, five kinds of polypeptide (1) (SEQ ID NO: 10to 14), each containing a plurality of these regions, were designed inthis Test Example. At the same time, an original SE36 polypeptide(polypeptide (2): SEQ ID NO: 1), and SE36 without the N-terminal region(negative SE36: SEQ ID NO: 15) as a negative control were designed.FIGS. 1-1 to 1-2 show the amino acid sequences of these polypeptides.FIGS. 1-3 to 1-9 (SEQ ID NO: 16 to 22) show the base sequences ofnucleotides encoding the amino acid sequences. FIG. 2 shows a schematicdiagram of the structures of five kinds of polypeptide (1), polypeptide(2), and negative SE36 polypeptide.

The method for preparing the polypeptides used in Test Example 1 isdescribed below. Although a method of preparing the polypeptide (2) isdescribed in detail, other polypeptides can also be produced in asimilar manner.

Construction of Polypeptide Expression System

The expression system of the polypeptide (2) (SEQ ID NO: 1) wasconstructed in the following manner.

The DNA base sequence of the full-length SE36 gene that had beentheoretically converted from Pf codons to Escherichia coli codons wasdivided into 8 fragments. For each divided fragment, a sense (+) strandand an antisense (−) strand were synthesized to obtain 16single-stranded DNA fragments in total (8 pairs), which were annealed togive 8 pairs of double-stranded DNA. These sequences were ligated toeach other to give a full-length of SE36 gene, from which an expressionvector was constructed.

In this operation, the basic procedure for cloning and ligation of thesynthetic DNA fragments was conducted in accordance with the method ofSambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd edition,Cold Spring Harbor Laboratory Press, 1989).

The above single-stranded DNA fragments were individually synthesizedusing an Applied Biosystems Model 392 DNA/RNA synthesizer (a product ofPE Co., USA). These synthesized fragments were purified byelectrophoresis on 10% (w/v) polyacrylamide (containing 50 mMTris-borate salt, pH 8.3, 1 mM EDTA, and 8M urea). Then, 20 pmoles ofthe + and − complementary strands of each purified DNA fragment weremixed, and then heated in a buffer solution (20 μl of 20 mM Tris-HCl, pH7.0, 50 mM NaCl, and 2 mM MgCl₂) at 85° C. for 5 minutes.

Further, the complementary regions of both the strands above wereannealed by lowering the temperature to 55° C. at a rate of 5° C./5minutes and then to 25° C. at a rate of 5° C./10 minutes using aZymoreactor II (a product of ATTO Co., Japan). After annealing, an equalamount of a buffer solution (20 mM Tris-HCl, pH 7.8, 10 mM MgCl₂, 5 mMdithiothreitol (DTT), 1 mM each of 4 species ofnucleoside-5′-triphosphate (NTP), and 3 units of T4 DNA polymerase) wasadded, and the mixture was kept at 4° C. for 5 minutes, 25° C. for 5minutes, and then at 37° C. for 120 minutes.

For construction of SE36 gene, the resulting double-stranded DNAfragments were individually digested with restriction enzymes KpnI andBamHI, and then cloned and multiplied with pBluescript II SK+ andEscherichia coli XL1-Blue. The base sequences of the above DNA fragmentsin each of the clones were determined by the dideoxy method, and 8clones covering the full length of SE36 gene were screened. Thesynthesized double-stranded DNA fragments of these 8 clones (8 pairs)were ligated to give a full length of SE36 double-stranded DNA.

In this operation, using a base sequence designed in such a manner thatthe amino acid sequence of the native Pf was not altered, therestriction enzyme sites for ligation were introduced to both ends ofeach pair of DNA. Subsequently, the full length of an SE36 gene wascloned with pBluescript II SK+, and then transfected to Escherichia coliXL1-Blue for proliferation. The base sequence was determined by thedideoxy method. The results are shown in SEQ ID NO: 10 in the SequenceListing.

Subsequently, the fragments of the above clone digested with restrictionenzymes NdeI and BamHI were inserted and ligated into the NdeI-BamHIcleavage sites of a plasmid pET-3a to construct a SE36 expression vectorpET-SE36. This expression vector was transfected to Escherichia coliBL21 (DE3) pLysS to give a transformant, Escherichia coli BL21 (DE)pLysS/pET-SE37, which was designated as Escherichia coli BL/SE36.

Expression and Purification of the Polypeptide

The Escherichia coli BL/SE36 obtained above was incubated on an LBmedium (Bacto-trypton 1% (w/v), Bacto-yeast extract 0.5% (w/v), and NaCl1% (w/v)) containing 50 μg/ml of ampicillin at 37° C. for 18 hours togive seeds. The seeds (50 ml) were inoculated on fresh LB medium (5 L)and incubated at 37° C. When the number of cells reached 1×10⁸/ml, IPTG(isopropyl-β-D-thiogalactopyranoside) was added at a final concentrationof 50 μg/ml, and further incubated at 37° C. for 3 hours. After theincubation, the mixture was centrifuged (5,000 rpm, 10 minutes) tocollect the cells. 3.2 g of cell paste was obtained. The paste wassuspended into 9.6 ml of an ice-cold lysis buffer solution (50 mMTris-HCl, pH 8.0, and 1 mM EDTA). Then, the procedures (1) to (6) wereconducted at 4° C. in the order as described.

(1) Sonication

The above cell paste was disrupted by treatment with ultrasonic waves(19.5 kHz, 50 W) 6 times for 20 seconds. The supernatant aftercentrifugation (15,000 rpm, 30 minutes) was collected and placed in abeaker of 20-ml volume.

(2) Salting-Out with Ammonium Sulfate (I)

To the supernatant in the beaker was added 2.37 g of (NH₄)₂SO₄ crystalswith stirring to achieve a saturation of 35% (W/W). The mixture wasfurther stirred for 30 minutes for salting-out. Subsequently, themixture was centrifuged (12,000 rpm, 10 minutes), and the supernatantwas discarded. The precipitate was suspended in 9 ml of an ice-coldammonium sulfate solution (a lysis buffer solution as described abovecontaining 1.1 M (NH₄)₂SO₄) at an ammonium sulfate saturation of 30%(w/w). The resulting suspension was centrifuged (12,000 rpm, 10 minutes)and the supernatant was discarded. The precipitate was suspended againinto 8.8 ml of a lysis buffer solution (50 mM Tris-HCl (pH 8.0), 1 mMEDTA, 50 mM 2-mercaptoethanol, 9 M urea, and 1% (w/v) TWEEN80(polysorbate 80)) and recovered. Half the volume (4.4 ml) of therecovered suspension was heated at 60° C. for 10 minutes, then againice-cooled and filtered through a 0.45-μm filter (a product ofMillipore, USA).

(3) Column Purification (I)

The filtrate was chromatographed on a column of SEPHACRYL S-300 (26/60)(gel filtration media) equilibrated with a GF buffer solution (50 mMTris-HCl (pH 8.0), 1 mM EDTA, 50 mM 2-mercaptoethanol, and 8M urea) (3.5ml/fraction; flow rate=0.3 ml/minute; 4° C.). Each of the fractions22-43 was subjected to SDS-polyacrylamide electrophoreses. Based ontheir migration patterns, the fractions 32-37 containing a large amountof SE36 protein were pooled. The remaining resuspended solution (4.4 ml)was also treated in the same manner as above, then combined with thepooled fractions above, and used in the subsequent operation (4).

(4) Column Purification (II)

The resulting pooled fractions were kept at room temperature, and(NH₄)₂SO₄ was added thereto with stirring at an amount of 0.093 g per mlof the pooled fractions to achieve a final ammonium sulfateconcentration of 0.7 M. On the other hand, an aqueous column with 13 mlof OCTYL SEPHAROSE (agarose beads, a product of Pharmacia Biotech) wasequilibrated with a 10-fold volume of an HIC buffer solution (a GFbuffer solution as mentioned above containing 0.7 M (NH₄)₂SO₄). Theammonium sulfate-adjusted pooled fractions were poured onto the columnat a rate of 0.5 ml/minute. Subsequently, the HIC buffer solution wasadded to the column until the absorbance decreased, and then forconfirmation, the column was eluted with a GF buffer solution that doesnot contain (NH₄)₂SO₄ to elute the adsorbed components. The fractionsnot adsorbed on the column were placed in a dialysis bag and dialyzedagainst 1 L of 20-mM Tris-HCl buffer solution (pH 8.0) (containing 1 mMEDTA) at 4° C. for 10 hours. During the dialysis, this outside solutionwas changed twice.

(5) Salting-Out with Ammonium Sulfate (II)

The dialysis bag after completion of the dialysis was further dialyzedagainst 0.3 L of a 50% (w/w) saturated (NH₄)₂SO₄ solution (containing 20mM Tris-HCl (pH 8.0) and 1 mM EDTA) at 4° C. for 10 hours to obtainproteins as a precipitate. The precipitate was collected bycentrifugation (12,000 rpm, 10 minutes) and suspended into 2 ml of GFbuffer solution.

(6) Column Purification (III)

The suspension obtained above was heated at 60° C. for 10 minutes andthen cooled back to 4° C. This was filtered through a 0.45-μm filter.The filtrate was chromatographed on a column of the above-mentionedS-300 (26/60) equilibrated with a GF buffer solution 2 (10 mM Tris-HCl(pH 8.0), 1 mM EDTA, 20 mM 2-mercaptoethanol, and 8 M urea) at a flowrate of 0.3 ml/minute. In the same manner as in the above item (3), eachfraction was applied to SDS-polyacrylamide electrophoresis to screenfractions of the SE36 protein, which were collected to pool 12 mlfraction. This fraction was added to a dilution buffer (10 mM Tris-HCl(pH 8.0), 1 mM EDTA, and 2 M urea) with stirring so as to give asolution of the SE36 protein at a concentration of 25 μg/ml. The dilutedsolution was dialyzed against 2 L of PBS (9 mM NaHPO₄, 3 mM NaH₂PO₄, and137 mM NaCl (pH 7.4)) at 4° C. for 10 hours. During this operation, theoutside solution was changed twice. Subsequently, the inside solutionafter dialysis was concentrated with CENTPREP 30 (centrifugal filterunit), and filtered through a Durapore 0.22 μm-filter (a product ofMillipore, USA) for sterilization to give 10 ml sterile specimencontaining 1 mg/ml of the SE36 protein. This was stored as a stocksolution for an SE36 vaccine at 4° C.

Determination of the Amino Acid Sequence

The amino acid sequence of the polypeptide (2) thus obtained wasdetermined by Edman degradation using an Applied Biosystems 473A proteinsequencer (a product of PE Co., USA). The polypeptide (2) was confirmedto have the amino acid sequence set forth in SEQ ID NO: 1.

Test Example 2

Using the polypeptides (1) (SE36-1 to SE36-5 polypeptides), polypeptide(2), and negative SE36 polypeptide as antigens, a test was performed inthe following manner.

Mice were immunized with each of the purified polypeptides (antigens),and induction of antibody titers was monitored. More specifically,thirty-five C57BL/6_tlr4KO mice were divided into 7 groups of 5 miceeach, and each group was inoculated with a mixture of one antigen withaluminium hydroxide gel and a K3 adjuvant by subcutaneous injection.FIG. 3 shows the results.

The immunizing amount was defined as 13 μg of aluminium hydroxide geland 50 μg of K3 per μg of the antigen. As indicated by an arrow in FIG.3, a second immunization was performed two weeks after the firstimmunization. Every week, the blood was sampled to obtain the serum, andanti-SE36-IgG antibody titers were determined by an ELISA assay. TheELISA assay was performed in the following manner.

Preparation of ELISA Plate

SE36 was diluted to 1 μg/ml with a coating buffer (pH 9.6, containing3.4 g of Na₂CO₃ and 5.7 g of NaHCO₃ in 1 L of distilled water). Thediluted SE36 was dispensed into each well of a 96-well MaxiSorpNUNC-Immuno plate (442404: Nunc) using 100 μl per well. The plate waswashed twice with a wash buffer (PBS(−) containing 0.05% TWEEN 20(polysorbate 20)), and then blocked with a blocking buffer (5% skim milkand PBS(−) containing 0.05% Tween 20) and incubated at 4° C. for 2hours. The blocked ELISA plate was washed with washing buffer 4 to 5times and stored at −20° C. until use.

Reaction with Test Sera

(a) Standard Serum

Plates with 12 (row)×8 (column) wells were used. A blocking buffer wasdispensed into each well of the first row in an amount of 150 μl, andinto each well of the subsequent rows in an amount of 100 μl. 1.5 μl ofa standard serum was placed into the wells of the first row, and theserum and the blocking buffer were mixed thoroughly (1/100 dilution). 50μl each well of the first row was transferred to the wells of theadjacent row and mixed thoroughly (1/3 dilution). This operation wasrepeated until the wells of the last row (whereby the serum can beserially diluted to 1/100, 1/300, 1/900, 1/2700, etc.).

(b) Test Sera

Each sample (serum) was diluted with the blocking buffer (to 1/100,1/500, 1/1000, etc.: the sample was diluted in such a manner that theO.D. values fell within the standard curve). The diluted sample wasdispensed into each well in an amount of 100 μl per well. The sample wasincubated at room temperature for 2 to 3 hours, or at 4° C. overnight,and then washed with the wash buffer 4 to 5 times.

Reaction with a Secondary Antibody

The secondary antibody (Anti-Mouse IgG (Goat Anti-Mouse IgG (H+L)))(1031-05: SouthernBiotech) was diluted with a blocking buffer to adilution factor. The resulting mixture was dispensed in an amount of 100μl per well and incubated at room temperature for 2 to 3 hours, or at 4°C. overnight, followed by washing with the wash buffer 4 to 5 times. Thedilution factor of the second antibody can be set as desired.

Detection

TMB (3,3′,5,5′-tetramethylbenzidine: Sigma: T8665-100ML), which is achromogenic substrate, was dispensed in an amount of 50 μl per well.When the sample had turned an appropriately dark blue, 1 mol/l sulfuricacid (Nacalai Tesque: 95626-06) was dispensed in an amount of 50 μl perwell. Detection was performed at 450 nm using a plate reader, andbackground was subtracted at 540 nm.

As shown in FIG. 3, all the polypeptides (1) (SE36-1 to SE36-5) providedhigher antibody titers than the polypeptide (2). In particular, SE36-3to SE36-5 induced high antibody titers. These results indicate that thepolypeptide (1) of the present invention, i.e., an improved SE36polypeptide, can induce an anti-SE36 antibody several ten to severalhundred times higher than the amount induced by the polypeptide (2).

Epitope Mapping

Epitope mapping was performed using the serum obtained in the 4th week.For the epitope mapping, the wells of plates were coated with fifteenpeptides set forth in SEQ ID NOS: 23 to 37 at a concentration of 0.03 mM(see FIGS. 1 to 9), and the ELISA assay was performed. FIG. 4 shows theresults. The results of epitope mapping indicate that the polypeptides(1) (SE36-1 to SE36-5) highly promote induction of an antibody to theN-terminal region that is important as a protective epitope.

More specifically, induction of the antibody to the N-terminal region ofa SE36 polypeptide by using the polypeptide (2) (original SE36polypeptide) as an antigen is as shown in the upper-left graph of FIG.4. The results of FIG. 4 show that the polypeptides (1), i.e., SE36-1 toSE36-5 (particularly SE36-3 to SE36-5) can remarkably enhance theinduction of the antibody to the N-terminal region of a SE36 polypeptide(epitope polypeptides 1 to 3 (polypeptides set forth in SEQ ID NOS: 23to 25)).

Test Example 3

As shown below, adjuvants (aluminium hydroxide gel, K3 (K-type CpGadjuvant), D35 (D-type CpG adjuvant), and sHZ (synthetic hemozoinadjuvant) were used in combinations, and their immunogenicity-enhancingeffects on the polypeptide (2) were evaluated.

(1) Enhancement of Antibody Induction Capacity by Combined Use ofAdjuvants

Immunization was performed using the polypeptide (2) alone as theantigen or using the polypeptide (2) with aluminium hydroxide gel, thepolypeptide (2) with K3, or the polypeptide (2) with aluminium hydroxidegel and K3. The test method was performed in the same manner as in TestExample 2. Four weeks after the first immunization, anti-SE36-IgGantibody titers were measured. Table 5 shows the results. The results ofFIG. 5 show that the combined use of aluminium hydroxide gel and K3 asadjuvants (indicated as “Alum+K3” in FIG. 5) can remarkably induce ananti-SE36 antibody as compared to using the antigen alone (indicated as“none” in FIG. 5), using only aluminium hydroxide gel as the adjuvant(indicated as “Alum” in FIG. 5), and using only K3 as the adjuvant(indicated as “K3” in FIG. 5).

(2) Enhancement of Antibody Induction Capacity by Combined Use ofAdjuvants

The present inventors further evaluated the enhancement ofimmunogenicity of the polypeptide (2) by adjuvants other than thecombination of aluminium hydroxide gel and K3. In this test, antibodytiters in test animals were evaluated over a long period of time. Thespecific test method is as shown below.

Twelve cynomologus monkeys were divided into 4 groups of 3 monkeys. Themonkeys were immunized by subcutaneous injection using the following asadjuvants for the polypeptide (2): aluminium hydroxide gel only;aluminium hydroxide gel and K3 (K-type CpG adjuvant); aluminiumhydroxide gel and D35 (D-type CpG adjuvant); and aluminium hydroxide geland sHZ (synthetic hemozoin adjuvant).

For the immunization, vaccines prepared by adding 500 μg of either K3 orD35, or 1.5 mM sHZ to the polypeptide (2) (10 μg) and aluminiumhydroxide gel (125 μg) were used. Immunization was performed at the timepoints indicated by arrows in FIG. 6 (day 0, day 22, and day 101). Ondays 0, 7, 14, 22, 28, 36, 42, 56, 73, 86, 101, 112, 140, 175, 205, 238,268, and 365, blood was drawn, and the serum was collected. Theanti-SE36-IgG antibody titers in the serum were measured by an ELISAassay. The ELISA assay was performed in the same manner as in TestExample 2. In Test Example 3, anti-monkey IgG (wholemolecule)-peroxidase, antibody produced in a rabbit (A2054: Sigma), wasused as a second antibody. FIG. 6 shows the results.

The results of this test show that compared to the use of aluminiumhydroxide gel alone, combined use of aluminium hydroxide gel with K3(K-type CpG adjuvant), D35 (D-type CpG adjuvant), or sHZ (synthetichemozoin adjuvant) can enhance the immunogenicity of the polypeptide(2), and that in particular, combined use of aluminium hydroxide gelwith K3 can provide high antibody titers and also maintain this effectover a long period of time.

Epitope Mapping

Epitope mapping was performed using the sera with the highest antibodytiters (indicated with an asterisk (*) in FIG. 6). For the epitopemapping, the wells of the plates were coated with fifteen peptides shownin FIGS. 1 to 9 (SEQ ID NOS: 23 to 37) (concentration: 0.03 mM) andsubjected to the ELISA assay. A comparison was made using one animalhaving the highest antibody titer in each group. The results show thataddition of K3 (K-type CpG adjuvant) can promote antibody titers to theN-terminal region (FIG. 7: circled portions).

The results of this Test Example suggest that addition of a TLR9 ligandadjuvant (K3 (K-type CpG adjuvant), D35 (D-type CpG adjuvant), or sHZ(synthetic hemozoin adjuvant)) can enhance the immunogenicity of thepolypeptide (2), and the preparation containing a TLR9 ligand adjuvantcan be used as a more effective malaria vaccine.

In the above Test Example, the vaccinated animals were all healthy, andno unusual weight loss; abnormality in behavior, excrement, orappearance; or death was observed. More specifically, safety of thevaccine used in this Test Example was confirmed.

Test Example 4

Squirrel monkeys were immunized with a combination of the polypeptide(2) with adjuvants (aluminium hydroxide gel and K3 (K-type CpGadjuvant)), and the protective effects against malaria parasite wereevaluated.

Seven squirrel monkeys were randomly divided into three groups. Each ofthe three groups of squirrel monkeys was immunized by subcutaneousinjection with polypeptide (2) and aluminium hydroxide gel (n=2);polypeptide (2), aluminium hydroxide gel and K3 (n=3) (K-type CpGadjuvant); or aluminium hydroxide gel and K3 (n=2) (K-type CpG adjuvant)twice at three-week intervals. The immunization was performed in such amanner that the total dosage of the components, i.e., (polypeptide (2)(10 μg), aluminium hydroxide gel (125 μg), and K3 (500 μg)), was 0.5 ml.After the immunization, a live malaria parasite was inoculated in aconcentration of 5×10⁸ via the femoral vein. The number of malariaparasite-infected red blood cells in each squirrel monkey was determinedevery day for 14 days.

FIG. 8 shows the results. In one of the monkeys (n=2) in the groupadministered with polypeptide (2) and aluminium hydroxide gel, and bothmonkeys (n=2) in the group administered with aluminium hydroxide gel andK3, the number of malaria parasite-infected red blood cells reached ashigh as about 40 to 50%, and these monkeys died ethically by day 9, asshown in FIG. 8. In contrast, in all three monkeys in the groupadministered with polypeptide (2), aluminium hydroxide gel, and K3(n=3), the number of malaria parasite-infected red blood cells was lowerthan 30%, and none of the monkeys in this group died.

These results show that compared to the use of aluminium hydroxide gelalone, combined use of K3 (K-type CpG adjuvant) with aluminium hydroxidegel can more effectively enhance the immunogenicity of the polypeptide(2) and inhibit the growth of the malaria parasite, thus preventing thedeath of infected subjects.

[Sequence Listing Free Text]

-   SEQ ID NO: 1 is the amino acid sequence of polypeptide (2).-   SEQ ID NO: 2 is the amino acid sequence of A₁.-   SEQ ID NO: 3 is the amino acid sequence of A₂.-   SEQ ID NO: 4 is the amino acid sequence of A₃.-   SEQ ID NO: 5 is the amino acid sequence of A₄.-   SEQ ID NO: 6 is the amino acid sequence of A₅.-   SEQ ID NO: 7 is the amino acid sequence of A₆.-   SEQ ID NO: 8 is the amino acid sequence of A₇.-   SEQ ID NO: 9 is the amino acid sequence of B.-   SEQ ID NO: 10 is the amino acid sequence of an SE36-1 polypeptide.-   SEQ ID NO: 11 is the amino acid sequence of an SE36-2 polypeptide.-   SEQ ID NO: 12 is the amino acid sequence of an SE36-3 polypeptide.-   SEQ ID NO: 13 is the amino acid sequence of an SE36-4 polypeptide.-   SEQ ID NO: 14 is the amino acid sequence of an SE36-5 polypeptide.-   SEQ ID NO: 15 is the amino acid sequence of a negative SE36-6    polypeptide.-   SEQ ID NO: 16 is the base sequence of a nucleotide encoding the    amino acid sequence of polypeptide (2).-   SEQ ID NO: 17 is the base sequence of a nucleotide encoding the    amino acid sequence of an SE36-1 polypeptide.-   SEQ ID NO: 18 is the base sequence of a nucleotide encoding the    amino acid sequence of an SE36-2 polypeptide.-   SEQ ID NO: 19 is the base sequence of a nucleotide encoding the    amino acid sequence of an SE36-3 polypeptide.-   SEQ ID NO: 20 is the base sequence of a nucleotide encoding the    amino acid sequence of an SE36-4 polypeptide.-   SEQ ID NO: 21 is the base sequence of a nucleotide encoding the    amino acid sequence of an SE36-5 polypeptide.-   SEQ ID NO: 22 is the base sequence of a nucleotide encoding the    amino acid sequence of a negative SE36 polypeptide.-   SEQ ID NO: 23 to 37 are the amino acid sequences of epitope mapping    polypeptides 1 to 15.

The invention claimed is:
 1. A polypeptide as set forth in formula (1):X₁-A-B-X₂-Y-X₃-(Y)n-X₄-(Y)n-X₅  (1) wherein X₁ represents the 1st to 7thamino acid residues in a polypeptide set forth in SEQ ID NO: 1; X₂represents the 73th to 177th amino acid residues of the polypeptide setforth in SEQ ID NO: 1; X₃ represents the 178th to 258th amino acidresidues of the polypeptide set forth in SEQ ID NO: 1; X₄ represents the259th to 289th amino acid residues of the polypeptide set forth in SEQID NO: 1; X₅ represents the 290th to 334th amino acid residues of thepolypeptide set forth in SEQ ID NO: 1; A represents an 8-mer repeatsequence contained in a 47-kd region of a SERA polypeptide of Plasmodiumfalciparum; B represents the amino acid sequence set forth in SEQ ID NO:9; Y represents any one selected from A-A, A-B, and B; and n representsan integer of 0 or
 1. 2. The polypeptide according to claim 1, wherein,in formula (1), Y is A-B or B.
 3. The polypeptide according to claim 1,wherein, in formula (1), A is an amino acid sequence selected from SEQID NOS: 2 to
 8. 4. The polypeptide according to claim 1, comprising theamino acid sequence set forth in one of SEQ ID NOS: 10 to
 14. 5. Apolynucleotide encoding the polypeptides of any one of claims 1 to 4.